The aim of this project is to define the molecular details controlling signal transduction in response to arginine during biofilm formation; we plan to define how this nutrient modulates c-di-GMP (3',5'- cyclic diguanylic acid) levels in Pseudomonas aeruginosa. This bacterium is an opportunistic human pathogen responsible for biofilm-mediated chronic infections; biofilms represents a sessile multicellular lifestyle difficult to be eradicated, which confers both antibiotic and host defence resistance to the encapsulated bacterial population. Nutrients may control intracellular levels of the second messenger C-di-GMP, which in turn trigger biofilm formation when accumulates. Among nutrients, arginine represents a strategic metabolite being at the crossroad of many metabolic processes (including carbon, nitrogen and ATP source) and also associated to chronic infections, biofilm/virulence and antibiotic resistance.
We recently found that P. aeruginosa is able to respond to environmental arginine by directly decreasing the intracellular levels of c-di-GMP via the molecular "antenna" named RmcA (Redox regulator of c-di-GMP). This multidomain membrane protein recognise extracellular arginine through a Venus Fly Trap (VFT) domain and transduces the environmental signal by a transmembrane and cytoplasmic portions containing PAS-LOV domains; their re-organization controls the downstream catalytic moiety composed by the diguanylate cyclases (GGDEF) and the phsphodiesterases (EAL) tandem, where the hydrolysis of c-di- GMP occurs.
The final goal of this proposal is to gain details on the mechanism of control of arginine on c-di- GMP hydrolysis both in terms of VFT activation and of PAS-mediated transduction of the periplasmic signal.
This mechanistic study will be useful to develop an approach to promote biofilm dispersion (where cells are more sensitive to traditional antimicrobial compounds) to finally treat chronic infection with more effective combined strategies.
Tuning of cyclic dinucleotides in prokaryotes (and also in eukaryotes) as a strategy to control crucial cellular fates is by now acknowledged by the scientific community; the idea that these second messengers control complex cellular re-organization by re-shaping the basal metabolism is also well-established. In light of this, understanding the molecular details controlling the communication between nutrients relevant to central metabolism and dinucleotide signalling is mandatory for the development of future strategies to control cell destiny.
For bacteria, a promising approach to target biofilm-mediated infections is to interfere selectively with the metabolic re-programming necessary to trigger biofilm formation via dinucleotide sensing; however, despite the promising results in vitro, up to now the direct targeting of the enzymes involved in c-di-GMP turnover was found to be ineffective and poorly selective on cells (Schirmer T et al., 2009; Krasteva PV et al., 2012; Sondermann H et al., 2012).
Nevertheless, the potential therapeutic impact deriving from the inhibition of biofilm via c-di-GMP in human diseases is huge being 65-80% of all infections in developed countries biofilm-mediated (NIH). In 2000, the Center for Disease Control and Prevention (CDC) announced biofilms and biofilms-mediated infections as two of seven major healthcare problems facing the medical community in the 21th century. Biofilms weighs on the healthcare and, more in generale, on public budget not only for infections (particularly the nosocomial and the chronic ones) but also for equipment damage, energy losses and product contamination. Related issues include deterioration of dental surfaces, contamination of surfaces in the food processing industry, water pipelines and the deterioration of air quality in ventilation and air handling systems (Bryers JD, 2008). The biofilm issue is strictly linked to the implantable devices, which, although their use and development is continuously growing, get contaminated more easily than native tissue (10,000 times less bacterial load); together with the problem of medical devices, bacterial biofilms on chronic wounds are a major cause of hospital-associated infections, with high morbidity and mortality (Khatoon et al., 2018).
The idea to tackle c-di-GMP issue from the environment represent a promising strategy to promote biofilm dispersal; the fact that biofilm-dispersed bacteria are more susceptible to traditional antibiotics than their sessile counterpart, suggests that a combined treatment "cue(s)+drug" could represent a future approach to interfere with biofilm success.
The main goals of this project are achievable, also considering the unique and complementary expertise of the RU members and the features and reputation of the Department of Biochemical Sciences, where the project will be carried out (see the next paragraph for details).